The overall objective of this project is to understand the cellular and molecular mechanisms responsible for the specification, patterning, and differentiation of internal organs during development. More specifically, how the elaborate network of blood vessels arises during vertebrate embryogenesis. Blood vessels are ubiquitous and vital components of all vertebrate animals, innervating and supplying every tissue and organ with oxygen, nutrients, and cellular and humoral factors. They have also become a subject of great clinical interest in recent years, particularly with the potential shown by antiangiogenic therapies for combating cancer. Many of our insights into mechanisms of blood vessel formation have come from developmental studies. The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make it particularly well suited for studying blood vessels. The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo. These features provide a tremendous advantage for studying vascular development- they permit observation of every vessel in a living animal and simple, rapid screening for even subtle vascular-specific mutants. Major aims of the laboratory include: (i) Developing new experimental tools and resources to enhance the zebrafish as an experimental model for studying vascular embryogenesis, (ii) Studying the molecular basis for arterial-venous differentiation, (iii) Studying the role of neuronal guidance factors in vascular guidance and patterning, (iv) Performing forward genetic analysis of blood vessel development using vascular-specific mutants. Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish An important aim of this project has been to develop new experimental tools for studying blood vessel formation in this organism. Previously, we devised a microangiographic method for imaging patent blood vessels in the zebrafish and used this method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish and http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We have also generated several different transgenic zebrafish lines expressing green fluorescent protein (GFP) in vascular endothelial cells (VEC), making it possible for us to visualize the blood vessel formation in intact, living embryos. We have developed methodologies for long-term multiphoton confocal timlapse imaging of the dynamics of blood vessel formation in these transgenic zebrafish. Our findings highlight the extremely dynamic, unexpectedly growth cone-like behavior of growing angiogenic blood vessels. Molecular Dissection of Arterial-Venous Development We have uncovered a molecular pathway regulating the acquisition of arterial-venous identity. Although the fundamental distinction between these two types of blood vessels has been appreciated for thousands of years, the fact that arterial and venous endothelial cells have distinct molecular and functional identities has only become apparent very recently, and the mechanisms responsible for establishing this identity have not been elucidated. We have now shown that sonic hedgehog (SHH), vascular endothelial growth factor (VEGF), and notch signaling act in series to determine arterial venous identity. We were able to do this by using different combinations of drug treatments, mutants, morpholinos, and mRNA injections to activate or repress the activity of each of these signals in vivo, either alone or in combination. Our surprising findings regarding the novel role of VEGF in arterial specification have been confirmed by a number of very recent publications describing a similar activity for VEGF in mice. We are now exploring additional factors that might participate in the pathway we have uncovered. Analysis of the Roles of Neuronal Guidance Factors in Vascular Patterning We have recently become interested in the role that well-known neuronal guidance factors might be playing in vascular guidance and vascular patterning. \We have uncovered novel Robo and Plexin receptors expressed in zebrafish blood vessels and examined their functional roles. We are studying a novel Robo receptor expressed in both neuronal and vascular tissues in zebrafish. Vascular specific expression of a dominant-negative truncated form of this gene in zebrafish embryos (via transient transgenesis) results in premature vascular sprouting, consistent with the pro-migratory activity of this construct in vitro. We are also studying a vascular-specific plexin gene. Targeted knock-down of this gene using antisense morpholine oligonucleotide injection results in aberrant pathfinding of trunk intersegmental vessels and other vessels. Further analysis of the functional roles and activities of both of these receptors, and their ligands, is in progress. Our work suggests that the mechanisms of axonal and vascular guidance and patterning share a great deal in common, including specific molecular guidance factors. Isolation and Analysis of Vascular-Specific Mutants Genetic dissection of vascular development and the molecular pathways that regulate it is an important ongoing goal of the UVO. We employ unbiased, forward genetic mutational screening approaches to identify, and then phenotypically and molecularly characterize, zebrafish mutants that affect the formation of the developing vasculature. We have already positionally cloned the defective genes from a number of previously identified vascular patterning mutants. violet beauregarde mutants have abnormal cranial vascular patterning and circulation resulting from defects in a zebrafish ortholog of the TGF-beta superfamily receptor acvrl1 (Roman et al., 2002). Defects in human acvrl1 cause Hereditary Hemorrhagic Telangiectasia type 2 (HHT), an inherited vascular disorder characterized by arterial-venous malformations with a high incidence of hemorrhage and stroke. Another mutation, kurzschluss, has defects in the posterior aortic arches caused by defects in a chaperonin expressed in the mesenchyme surrounding the arch vessels, that may be involved in regulating cytoskeletal assembly. We have also performed new screens for vascular specific mutants using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. We identified 11 new vascular mutants in a pilot screen of haploid progeny of ENU mutagenized animals that was performed with the Dawid and Liu labs (members of these labs independently screened for hematopoietic and neuronal defects). We have already determined the molecular basis for one mutant, uncovering a gene potentially involved in VEGF signal transducction. L ike animals in which VEGF itself is directly targeted, these mutants display defects in arterial endothelial differentiation. Molecular and phenotypic characterization of other mutants from this pilot screen is in progress. We have recently initiated a larger-scale F3 diploid screen of ENU mutagenized animals (to be carried through with members of the Dawid and Chitnis labs) to screen for mutants affecting both intersegmental vessel formation and later vascular patterning events that cannot be examined in haploid animals. Our experience suggests that these ongoing mutant screens should continue to yield a rich harvest of novel vascular-specific mutants and bring to light new pathways regulating the specification, differentiation, and patterning of the developing vertebrate vasculature.